Use of starch for transdermal applications

ABSTRACT

The invention relates to the use of substantially fully destructurized starch for transdermal applications in humans and animals, in particular the use of solid particles such as implants. The invention further relates to the implants manufactured of substantially fully destructurized starch, in addition to methods for manufacturing the implants.

This application is a 371 of PCT/NL95/00313, filed Sep. 20, 1995.

The present invention relates to the use of starch for transdermalapplications. The invention relates in particular to transdermalapplications of solid products made from starch, such as implants. Theinvention relates more particularly to solid products produced byextruding and/or injection moulding raw materials based on starch and/orcontaining starch, which products are intended for transdermalapplications. The invention further relates to the implants manufacturedfrom starch, which may or may not be kinetic and may be hollow or solid.

Particular medicines, vaccines and the like may be introduced in thehuman or animal body in the form of implants. In order to administerimplants (usually in solid form), ordinarily either a surgical incisionis made through which the implant can be placed or a trocar is used,with or without mandrel.

In the first method (e.g. Endocons liquid-containing implant, asdescribed in PCT/US93/04666), a local anaesthetic must first be givenand one or more sutures made after the implant has been administered.This is a time-consuming and costly operation.

In the second method the trocar is pricked through the skin and theimplant is pushed, optionally using the mandrel, into the tissues(subcutaneously/intramuscularly or in other organs). When an implant(for instance Crest-star™, Intervet, Netherlands) has to be arrangedunder the skin of the auricle of cattle., there is a danger of thecartilage present under the skin being damaged. Such damage increasesthe risk of inflammations and can adversely affect the release of theactive substances. There is moreover a danger, when the animal makes anunexpected movement, of piercing right through the auricle with thetrocar and even injuring the hand with which the auricle must be held.In the case a trocar with mandrel is used there is moreover a chance ofdamaging vulnerable implants (which contain antigens, hormones or otheractive substances and which are sold for instance in the form ofpellets), so that the desired pattern of release is no longer certain.

In addition to by means of surgical incisions and trocars, it is also aknown method to introduce implants into animals by means of ballisticbiodegradable bullets such as for instance the commercially available"Biobullets" from Ballistivet or those described in U.S. Pat. No.3,982,536, U.S. Pat. No. 3,616,758 and PCT/AU87/00091. The drawback ofBallistivet's Biobullets is their size (diameter 6.7 mm) and the factthat they contain up to 5% of the non-biologically degradable HydroxyPropyl Cellulose (HPC). As a result, pieces of tissue have to be cutaway during slaughter of the cattle treated therewith (the Biobulletsare too coarse for use with other animal species or humans).

The object of the invention is to provide a transdermally applicableproduct, with which one or more active ingredients can be introducedinto the human or animal body in simple and responsible manner.

Understood in this patent-application by "transdermal application" isany use wherein the relevant product (containing the active ingredient)is introduced into the body through the skin and/or mucous membranes.This does not therefore include uses wherein only the active ingrediententers the body. Subcutaneous, intramuscular, intrapleural,intraperitoneal and optionally intravenous administering can beenvisaged here.

By "active ingredient" is meant in the broadest sense any material whichmust be introduced into the human or animal body. It not only comprisestherapeutic, diagnostic of prophylactic compounds and compositions butalso for instance identification chips and the like.

According to the invention it has now been found that products which areat least partly manufactured from substantially fully destructurizedstarch, particularly implants, are very suitable as vehicle forintroducing active ingredients into the human or animal body intransdermal manner.

The term "substantially fully destructurized starch" means that thestarch is practically fully released from the cells and granules inwhich it is naturally found. In practice this means that native starchis treated such that the cells and granules are disrupted as fully aspossible whereby the starch is released.

It follows in fact from the literature that it is not self-evident thatstarch is suitable for transdermal use. Starch in nativenon-destructurized form has already been used for a long time forintended or unintended transdermal applications. It is thus known toadminister chemically modified starch (called "Hydroxy Ethyl Starch" or"HES") in dissolved state intravenously as plasma-substitute in case ofshook. To this end HES with a substitution degree of 70% is injectedinto the vein. The disadvantage of this product is that the starch isonly partly broken down by the body. A part is excreted via the urine,another part via the bile, but a last part remains in the circulationfor weeks and is then slowly deposited in the reticuloendothelialsystem, particularly that of the skin. This causes severe itching andpruritus (W. Jurecka et al., Arch. Dermatol. Res. 285: 13-19 (1993)).

Starch powder is further used as moisture-absorbing agent in surgicalgloves. A part of this powder may wholly unintentionally enter theoperation wound in specific cases. This is also a case of transdermalapplication of starch, albeit unintended. Once it has entered the bodythe starch can have adverse effects there, such as the occurrence ofinflammations and granulomas when native starch granules from operatinggloves enter the wound, for instance the intestines. Deutsch, M.,Gynecol. Obstet. Invest. 22(2), 110-112 (1986) describes a seriousperitonitis caused by such starch granules. Native starch can furthergive rise to sensitization of the skin (Fisher, A. A., Cutis 38(5),307-308 (1986)) or even fistula formation (Peters, E. J. Oral Pathol.15(8), 454-458 (1986)).

It has now therefore been found according to the invention that onlywhen substantially fully destructurized is the starch accessible in thebody to amylases, which ensure a rapid and completed gradation, wherebythe undesirable side-effects of starch in the body known from theliterature can be prevented.

According to the invention it has further been found that thissubstantially fully destructurized starch causes no toxicity phenomenasuch as allergy, inflammations, irritations and granuloma formation whenadministered (intentionally or unintentionally) transdermally. This willbe further illustrated in the accompanying examples.

It had not been determined prior to the present invention that starch insubstantially fully destructurized form, in contrast to native starch,is suitable to be administered transdermally as a solid product.

Substantially fully destructurized starch is per se known. It isdescribed for instance in European patent application 282.451 and istherein designated as "destructurized starch". However, no transdermalapplications are known from this publication.

The international application WO-92/15285 does however describe in agreat number of examples the manufacture of different forms ofmedication using destructurized starch. The object of the inventiondescribed in this patent application is however to provide forms ofmedication with controlled release. Although a number of transdermalforms of medication is mentioned in the description and claims, not asingle transdermal application is further described in the examples.Moreover, in vivo tests were not carried out with any of the mainly oralforms of medication described in the examples, whereby it is notpossible to deduce the in vivo effect of such forms of medication on thebasis of the description.

Substantially fully destructurized starch can be manufactured indifferent ways, for instance as described in EP-282.451. Thispublication describes a method for the destructurizing of starch bymeans of high temperature and/or high pressure, for instance byextruding native starch at a temperature of about 100-200° C.,preferably 140-190° C., most preferably 160-185° C. at a pressure of 0to 150 bar.

The injection moulding of substantially fully destructurized starch isalso described for instance in GB-2 190 093 and EP-304.401. Thepressures exerted on the starch during these injection mouldingprocesses lie between 300 and 3000 bar, preferably between 700 and 2200bar.

Many other destructurizing methods are of course conceivable for theobject of the invention, assuming that the destructurized starchobtained meets the requirements of a rapid degradation in the body andthe absence of the aforementioned undesired side-effects.

Preferably however the above mentioned extrusion/injection mouldingtechnique is used for destructurizing starch. The advantages ofextruding and injection moulding as method of destructurizing are thelow cost and the methods of performing these techniques under GoodManufacturing Practice (GMP).

The use according to the invention of starch which is substantiallyfully destructurized has a large number of applications which will bedescribed in further detail hereinbelow and form part of the presentinvention.

In one particularly advantageous embodiment of the invention the productmanufactured at least partly from substantially fully destructurizedstarch is an implant.

Such implants can be produced by means of per se known injectionmoulding techniques. The starch, for instance in the form of agranulate, is processed in the injection moulding machine to the desiredproduct. The destructurizing then also takes place in the injectionmoulding machine. Granulates which can serve as raw material formanufacture of the transdermal forms of medication according to theinvention are for instance Commercially available. An example of agranulate is SUNPEARLS™ from AVEBE (Foxhol, Groningen, The Netherlands).

Very specific extrusion/injection moulding parameters are preferablychosen to reduce the toxicity of the starch in the end products(implants). By varying the pressure, temperature, cycle time, amount ofwater and the like it can be ensured that the extruded end product doesnot become toxic. This is further illustrated in the examples.

Implants according to the invention for instance have the shape ofbullets. In veterinary medicine it is known to introduce inoculants andother transdermally administrable medication, hormones or prophylacticsubstances into the animal by means of kinetic implants ("bullets").Such implants can for instance be administered with an instrumentspecifically designed for this purpose which is described in theNetherlands patent application 92.00844. By manufacturing such bulletsaccording to the invention from substantially fully destructurizedstarch, problems such as inflammations and irritations in the animal canbe avoided.

The bullets may be hollow or solid depending upon their application.Hollow bullets can be filled with the material to be administered. Solidbullets are however manufactured from destructurized starch mixed withthe active ingredient. The usual additives such as albumins, gelatine,sugars, polylactic acid, copolymers with glycol acid, polyethyleneglycol (PEG) etc. can be added to the starch. This usually takes placeprior to the run through the injection moulding machine, for instance inorder to facilitate the processing of the starch in the machine or toprovide the end product with particular properties.

In principle it is possible to give the bullets any conceivable shapeand size. For pigs however are recommended bullets with an outerdiameter of 3 mm, a wall thickness of 0.35 mm and a length of 15 to 30mm. For cattle can be envisaged bullets with an outer diameter of 4.5mm, a wall thickness of 0.5 mm and a length of 45 mm. With regard to theshape a conventional bullet shape (FIG. 1A) can be envisaged but a shapewith a sharp tip and a ridge (FIG. 1B) is also possible. The sharp tipensures easy piercing of the skin, while the ridge ensures a maximumbraking effect once the skin has been penetrated. As a result theinjection depth can be controlled better.

The injection depth of the bullets can be influenced by differentparameters. The total weight of the filled bullet, the speed imparted tothe bullet, the shape of the tip and the amount of spin given theretoare therefore of importance. In this latter case applies that the morespin, the more unstable the bullet becomes when entering the tissue,whereby it will begin to tilt more quickly and will then be slowed downmore quickly, whereby it penetrates less deeply. The position of thecentre of gravity of the implant also determines the speed of tiltingafter penetration of the tissue, and thus the injection depth.

By means of a transparent tissue model of gelatine it has been foundthat bullets according to the invention, when they are introduced withan instrument such as described in NL-92.00844, if they should happen tofragment due to the presence of external damage, do leave fragmentsbehind but do not cause damage to the surrounding tissue. The bulletsaccording to the invention are therefore very safe.

The bullets do not have to be introduced with a special instrument suchas described in NL-92.00844 but they can also be introduced surgically,via a needle (trocar) with mandrel or in any other manner.

BRIEF DESCRIPTION OF THE DRAWINGS

In addition, it is possible in particular applications to push speciallydesigned bullets through the skin without any auxiliary means. Normally,trocars or injection needles are often used to introduce implants. Thedrawback hereof is that they generally have a very sharp tip with aradius of 0.01 mm or less and moreover possess two cutting faces. Theycan hereby cause damage to for instance the cartilage lying under theskin, such as in an auricle. According to the invention it has now beenfound that for pushing through the skin without auxiliary means theradius r of the tip of the implant is preferably smaller than 0.5 mm,more preferably smaller than 0.25 mm, most preferably between 0.03 and0.1 mm. The radius is independent of the angle (see FIG. 2) which thetangents of the side surfaces of the tip form with each other. To makethe tip as strong as possible it is recommended from a technicalviewpoint to make the said angle smaller than 30°, for instance between45° and 60°, to maximum of 120°. When technically feasible the angle mayof-course also be smaller than 30°. With a thus designed implant it ispossible to pierce thin skin of 0.1 to 1.5 mm thickness, depending onthe type of animal. Such thin skin is for instance found on the insideand outside of the auricle or adjacently of the anus of, for instancebut not exclusively, cattle, pigs, sheep and goats.

The sharpness of such bullets is not sufficient to pierce normal skin offor instance 3.0 to 3.5 mm thickness in the case of cattle, nor iscartilage damaged thereby, a danger which certainly does exist with theusual trocars. This is a great advantage, because damaged cartilage canresult in inflammations. The release of active ingredients from thebullet or the implant can also be adversely affected by damagedcartilage.

The active substances and other products which can be incorporated inthe hollow or solid bullets are very diverse. Herein can be envisagedmedicines, antigens, sex hormones for influencing the cycle in cattle,birth inducing medication for pigs, vaccines and even identificationchips.

The invention further provides a method for filling the bullets with anaqueous solution of for instance an active substance and thereafterfreeze-drying the active substance, whereby it remains behind in thebullet. In principle a bullet will dissolve as soon as it is filled withwater or an aqueous solution of an active ingredient. According to theinvention however a method is now provided wherein the bullet is frozenfor a time at for instance -20° C. and the aqueous solution of theactive substance, which may or may not be in gel form, is cooled for atime to for instance 4° C. After the bullet is filled with the solutionthe filled bullet is frozen again for a time whereafter it isfreeze-dried. In this manner the water disappears and the activesubstance remains behind in the bullet.

During production of the bullets various substances can be added, forinstance with a anaesthetic action (such as lidocaine) or with anantiseptic action (for instance iodine) or a colorant (such as forinstance methylene blue). Administering of the bullets to the animalscan be made easier, respectively more pleasant with these substances.

The implants according to the present invention administered forinstance using the device as described in Netherlands patent application92.00844 are further found to have an adjuvant action. The effectivenessof a good adjuvant depends on its ability to initiate an immune response(Michel Jolivet, in "Les adjuvants, ou comment doper les vaccins et lesysteme immunitaire", Biofutur, September 1989, p 45-52). It iscustomary to use adjuvants in vaccines, particularly in the case ofthose antigens which are poorly immunogenic in themselves. Adjuvants aretherefore always stimulating substances, which are in principleundesirable. Examples are aluminum hydroxide, polysorbates, soapysubstances like saponins (Quil A), Freunds complete or incompleteadjuvant and the like. In principle the presence of such substances inthe body is always undesirable. By using a needle-free method ofadministering the implants according to the invention it is in principlepossible to vaccinate with less or no addition of adjuvant. This resultsin cheaper production, fewer side effects and more predictable tissuereactions.

Starch consists of two different types of molecule, that is, amylose,the unbranched (1→4) polymer of glucose, and amylopectin, a (1→4)polymer with (1→6) branches. It has been found that during coolingamylose can group itself round free fatty acids and other polar and/oramphoteric substances such as lecithins. These groupings formcrystalline structures ("resistant starch") which are not enzymaticallydegradable, or only slowly. According to the invention it has now beenfound that these "resistant starch" structures do not result in negativephenomena, such as granuloma formation etc. This is very unexpected.

However, in order to avoid amylose being able to form such crystals, anamylose-free starch, the so-called "waxy maize starch," can be chosen asraw material for the starting granulate. This enhances the generalsolubility and total degradability.

Experiments which have resulted in the present invention havedemonstrated that the substantially fully destructurized starch isenzymatically degraded easily and quickly without toxic side effects.This will be shown in the examples.

The invention also relates to the use of substantially fullydestructurized starch as additive in other products intended fortransdermal application.

The present invention will be further elucidated with reference to theaccompanying examples which are not intended to limit the invention inany way but are only given by way of illustration.

EXAMPLE 1 Manufacture of Substantially Fully Destructurized Starch

The substantially fully destructurized starch according to theinvention, which is used in the following examples, was manufactured byextruding commercially available pure native starch, to which knownbio-compatible additives such as fat and lecithin and/or polyethyleneglycol and/or (water-soluble) hydrolyzed gelatine were added, at 160° C.and 150 bar according to the method described in EP-304.401 orEP-282.451. The extruded material was subsequently granulated bydividing the product leaving the extrusion device into particles ofseveral millimeters.

EXAMPLE 2 Manufacture of Starch Implants

Bullet-shaped implants according to the invention were manufacturedstarting from a granulate of the starch manufactured in example 1 in themanner described in EP-304.401 or EP-282.451. Use was made herein of aso-called hot-runner mould. The implants had a conventional bullet shapewith a diameter of 3 mm, a wall thickness of 0.35 mm and a length of 17mm.

EXAMPLE 3 Cytotoxicity Test

In order to determine the cytotoxicity of the implants of example 2, thesituation in the human body was imitated in a flow cell system, startingfrom the official ISO-protocol of ISO 10993. For this purpose a flow ofa liquid tissue culture medium with a determined flow speed was createdin a cell. The implants were placed in the system, whereby theygradually dissolved in the culture medium. On the outflow side of theflow cell a quantity of medium (so-called "extracts") were collectedover a number of time intervals. These extracts were subsequently placedin contact with fibroblasts, whereby the influence of the extracts onthe fibroblasts could be determined.

The tissue culture medium (extraction medium) was Minimal EssentialMedium (MEM), to which 10% foetal calf serum was added. As blank wasused an extract of this medium which had not been in contact with theimplants. As positive control an extract of RIVM-positive Latex wasused. The negative control was an extract of USP-negativeUHMW-polyethylene.

Prior to placing in the cell the implants were sterilized for 3 hours at50-53° C. in ethylene oxide and subsequently degassed for at least 48hours.

A flow of 0.025 ml per hour per product was generated in the flow cellsystem. In the cell were placed 20 bullets manufactured fromsubstantially fully destructurized starch. This is a total of 1 gstarch. The total rate of flow therefore amounts to 0.5 ml per hour. Anextraction volume of 12 ml was collected at 37° C. in each case at timeintervals of 24 hours. The estimated rate of flow of tissue liquidsthrough muscle tissue is 0.1 ml per gram tissue per minute. 6 ml pergram tissue thus passes per hour. A higher rate of flow automaticallyresults in a more rapid freshening of the liquid and a more rapidremoval of possible toxic substances. The liquid flow will consequentlybe greater in the body than in the flow cell used here. An extra safetymargin is incorporated by selecting a rate of flow for the cytotoxicityassay which is lower than the physiological rate of flow. Concentrationswhich are still just toxic in the test will no longer be so in the humanor animal body.

The extracts and dilutions thereof were tested for toxicity by bringingthem into contact with a monolayer of human fibroblasts of the typeL929, which were cultured to 80-100% confluence. After exposure to theextract the cells were examined microscopically for cytotoxic effects,such as the presence or absence of a monolayer, inhibition of cellproliferation (by means of a cell count), intracellular granulation,cellular swelling, and the percentage of cellular lysis was recorded.The results were determined in comparison with the negative control.

The results were classified as follows:

0-20% affected cells: evaluation 0, non-toxic;

20-50% affected cells: evaluation 1, slightly toxic;

50-70% affected cells: evaluation 2, mildly toxic;

70-90% affected cells: evaluation 3, moderately toxic;

90-100% affected cells: evaluation 4, very toxic;

The evaluations 0, 1 and 2 were still acceptable.

The results are shown in the table below.

                  TABLE 1                                                         ______________________________________                                                       Granulation                                                                             Growth in-                                                                             %    Morpho-                                Extract                                                                             Monolayer                                                                              evaluation                                                                              hibition Lysis                                                                              logy                                   ______________________________________                                        after 72                                                                             95%     2         45%      <5%  50%                                    hours                                  coil                                                                          shaped                                 1:1 di-                                                                             100%     0         18%      <5%  normal                                 lution                                                                        1:3 di-                                                                             100%     0         20%      <5%  normal                                 lution                                                                        1:7 di-                                                                             100%     0          0%      <5%  normal                                 lution                                                                        negative                                                                            100%     0          0%      <5%  normal                                 control                                                                       positive                                                                            partial  4         95%      --   cells                                  control                                                                             release                          coil-                                                                         shaped                                 ______________________________________                                    

The above shows that the products according to the invention are nottoxic.

EXAMPLE 4 Irritation Test

In this example is examined whether the implanting of bulletsmanufactured from substantially fully destructurized starch according tothe invention causes irritations in laboratory animals.

Extracts were taken from the implants manufactured in example 2 byextracting four grams of test material for 72 hours at 50° C. with 20 ml0.9% sodium chloride or cotton seed oil. Control solutions without thetest material were prepared in similar manner.

Three healthy rabbits free of significant skin impurities were used astest animals for each pair of extracts (test and control). The animalswere accommodated separately, were fed daily and had ad libitum accessto water. Prior to injection the hair on the back and sides of eachrabbit was cut short. Exactly 0.2 ml of the test extracts was injectedintracutaneously at four separate locations on the left side of the backof each animal while 0.2 ml control solution was injected at fourlocations on the right side. The injection locations were examined forerythema and oedema 24, 48 and 72 hours after injection and evaluated inaccordance with table 2. The average tissue reaction to the extract ofthe test object was compared to the control, which resulted in theirritation index.

Table 2 shows a survey of the evaluation of the intracutaneous response,and tables 3 and 4 show the results of the experiment. From theseresults there follows an irritation index of 0 for both the extractswith 0.9% NaCl and those with cotton seed oil. The implant according tothe invention does not therefore exhibit intracutaneous toxicity.

                  TABLE 2                                                         ______________________________________                                        Evaluation of intracutaneous response                                         ______________________________________                                        Erythema formation (ER):                                                      Evaluation 0: no erythema                                                     Evaluation 1: mild erythema                                                   Evaluation 2: well defined erythema                                           Evaluation 3: moderate erythema                                               Evaluation 4: serious erythema to mild scab forming                           Oedema formation (OE):                                                        Evaluation 0: no oedema                                                       Evaluation 1: mild oedema                                                     Evaluation 2: well defined oedema                                             Evaluation 3: moderate oedema; about 1 mm thick                               Evaluation 4: serious oedema; more than 1 mm thick                            ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Results of intracutaneous toxicity test with 0.9% sodium                      chloride in sterile demineralized water as extraction                         medium                                                                                     24 hours                                                                              48 hours  72 hours                                                    ER   OE     ER     OE   ER   OE                                  ______________________________________                                        TEST                                                                          Rabbit 1                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 2                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 3                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Control                                                                       Rabbit 1                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 2                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 3                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Results of intracutaneous toxicity test in cotton seed                        oil as extraction medium                                                                   24 hours                                                                              48 hours  72 hours                                                    ER   OE     ER     OE   ER   OE                                  ______________________________________                                        TEST                                                                          Rabbit 1                                                                              location 1 1      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 2                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 3                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Control                                                                       Rabbit 1                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 2                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               Rabbit 3                                                                              location 1 0      0    0    0    0    0                                       location 2 0      0    0    0    0    0                                       location 3 0      0    0    0    0    0                                       location 4 0      0    0    0    0    0                               ______________________________________                                    

EXAMPLE 5 Allergy Test

In order to examine whether the implants according to the inventioncaused allergic reactions a sensitization test was carried out.

The implants for testing were sterilized at 50-53° C. with ethyleneoxide and degassed for at least 48 hours.

A quantity of 4 grams test material was covered with 20 ml extractionmedium (sodium chloride in sterile demineralized water and cotton seedoil) and extracted at 70° C. for 24 hours. The control solutions(extraction liquid without implant) were prepared in a correspondingmanner.

Used as laboratory animals were albino guinea pigs of both sexes, with aweight of about 350 grams. For the testing of 1 extract 10 guinea pigswere treated with test material and 5 animals served as control group.

The guinea pigs were prepared by using electric clippers to remove thehair from an area of about 4×6 cm on the back above the dorsal capsulararea at least 24 hours prior to the test. For all local applications awoven bandage of 1×2 cm was saturated with the extract for testing (orthe control) and the bandage was applied to the shaven area under asealing bandage (surgical tape) for 6 hours. The control animals weretreated in similar manner with only the extraction liquids. Thisprocedure was repeated after one and after two weeks.

14 days after the last (third) induction application all test andcontrol animals were brought into renewed contact with the testmaterial. For this purpose bandages saturated with test extract wereapplied to untested areas of each animal. After 6 hours the bandageswere removed. 24 and 48 hours after removal of the bandages the animalswere shaven again if necessary and the reaction was evaluated. Theresults are shown in table 5. Each dermal inflammation response at thetest locations which was significantly greater than that observed undercontrol conditions was considered proof of an allergic reaction.Evaluations of one or more in the test group are generally seen as anindication of sensitization provided the control animals displayedevaluations of less than 1.

The evaluation of the allergic reaction was based on the same criteriaas stated in table 2. Tables 5 and 6 show the results respectively ofextracts of 0.9% sodium chloride in sterile, demineralized water andcotton seed oil.

None of the tested animals displayed a significant sensitization.

                  TABLE 5                                                         ______________________________________                                                    24 hours        48 hours                                                      ER   OE         ER    OE                                          ______________________________________                                        TEST                                                                          Animal 1      0      0          0   0                                         Animal 2      0      0          0   0                                         Animal 3      0      0          0   0                                         Animal 4      0      0          0   0                                         Animal 5      0      0          0   0                                         Animal 6      0      0          0   0                                         Animal 7      0      0          0   0                                         Animal 8      0      0          0   0                                         Animal 9      0      0          0   0                                          Animal 10                                                                    Average test  0      0          0   0                                         Control                                                                       control 1     1      0          0   0                                         control 2     0      0          0   0                                         control 3     0      0          0   0                                         control 4     0      0          0   0                                         control 5     0      0          0   0                                         control 6     0      0          0   0                                         average control                                                                             0.167  0          0   0                                         ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                                    24 hours        48 hours                                                      ER  OE          ER    OE                                          ______________________________________                                        TEST                                                                          Animal 1      0     0           0   0                                         Animal 2      0     0           0   0                                         Animal 3      0     0           0   0                                         Animal 4      0     0           0   0                                         Animal 5      0     0           0   0                                         Animal 6      0     0           0   0                                         Animal 7      0     0           0   0                                         Animal 8      0     0           0   0                                         Animal 9      0     0           0   0                                          Animal 10                                                                    Average test  0     0           0   0                                         Control                                                                       control 1     0     0           0   0                                         control 2     0     0           0   0                                         control 3     0     0           0   0                                         control 4     0     0           0   0                                         control 5     0     0           0   0                                         control 6     0     0           0   0                                         average control                                                                             0     0           0   0                                         ______________________________________                                    

EXAMPLE 6 Implantation Test

The implant according to the invention was sterilized with ethyleneoxide. HMWPE (high molecular polyethylene) strips of 1×10 mm were usedas control.

The backs of 3 albino rabbits of no more than 2.5 kg were shaven. Loosehair was removed. The paravertebral muscles were subsequentlyanaesthetized. 4 test samples were implanted in the right-handparavertebral muscle. 4 samples of the control plastic were implanted inthe left-hand paravertebral muscle. Three days after implantation therabbits were sacrificed and the samples and controls removed with thetissue surrounding them. The samples were embedded in paraffin or GMA,depending on the hardness of the sample material. The body reaction wasquantified using light microscopic observations. The results wereevaluated as shown in tables 7, 8, 9, 10. Understood by inflammationtype cells are macrophages, poly-mononuclear cells, lymphocytes,eosinophil cells, plasma cells and giant cells. The results are given intables 11 and 12.

                  TABLE 7                                                         ______________________________________                                                Degree of fibrosis                                                    ______________________________________                                                Evaluation 0: none observed                                                   Evaluation 1: up to 0.5 mm                                                    Evaluation 2: 0.5 to 1.0 mm                                                   Evaluation 3: 1.0 to 2.0 mm                                                   Evaluation 4: more than 2.0 mm                                        ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                                Changes in tissue morphology                                          ______________________________________                                                Evaluation 0: no changes                                                      Evaluation 1: slight changes                                                  Evaluation 2: mild changes                                                    Evaluation 3: moderate changes                                                Evaluation 4: serious changes                                         ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Presence of inflammation cells                                                ______________________________________                                        Evaluation 0:                                                                            no inflammation cells                                              Evaluation 1:                                                                            a number of inflammation cells                                                at contact location with sample                                    Evaluation 2:                                                                            various inflammation cells                                                    at contact location with sample                                    Evaluation 3:                                                                            many inflammation cells mainly                                                at contact location with sample                                    Evaluation 4:                                                                            many inflammation cells spread over the                                       whole area                                                         ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                                  Degree of necrosis                                                  ______________________________________                                                  Evaluation 0: none                                                            Evaluation 1: slight                                                          Evaluation 2: mild                                                            Evaluation 3: moderate                                                        Evaluation 4: serious                                               ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Implantation after 3 days                                                                                inflam-                                                           tissue      mation                                                      fibrosis                                                                            morphology  cells   necrosis                                   ______________________________________                                        TEST EXAMPLES                                                                 rabbit 1:                                                                     location 1 1       0           1     0                                        location 2 1       1           1     0                                        location 3 1       1           2     0                                        location 4 1       1           1     0                                        animal 2:  1       1           1     0                                        location 1                                                                    location 2 1       2           1     0                                        location 3 1       1           2     0                                        location 4 1       1           1     0                                        animal 3:  1       1           1     0                                        location 1                                                                    location 2 1       1           1     0                                        location 3 1       1           1     0                                        location 4 1       1           1     0                                        Average    1.0     1.0         1.2   0.0                                      CONTROL EXAMPLES                                                              rabbit 1:                                                                     location 1 1       9           1     9                                        location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        animal 2:  1       0           1     0                                        location 1                                                                    location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        animal 3:  1       0           1     0                                        location 1                                                                    location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        Average    1.0     0.0         1.0   0.0                                      ______________________________________                                    

The average evaluation was 1.2, which does not represent a significantreaction.

In the same manner a bodily reaction was evaluated two weeks afterimplantation. The results thereof are shown in tables 12 and 13. Theaverage body reaction evaluation was 1.0, which was likewise notsignificant.

                  TABLE 12                                                        ______________________________________                                        Implantation test after 2 weeks                                                                          inflam-                                                           tissue      mation                                                      fibrosis                                                                            morphology  cells   necrosis                                   ______________________________________                                        TEST EXAMPLES                                                                  rabbit 1:                                                                    location 1 1       2           2     0                                        location 2 1       2           2     0                                        location 3 1       2           2     0                                        location 4 1       2           2     0                                        animal 2:  1       0           1     0                                        location 1                                                                    location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        animal 3:  1       0           1     0                                        location 1                                                                    location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        Average    1.0     0.7         1.3   0.0                                      CONTROL EXAMPLES                                                              rabbit 1:                                                                     location 1 1       0           1     0                                        location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     9                                        animal 2:  1       0           1     0                                        location 1                                                                    location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        animal 3:  1       0           1     0                                        location 1                                                                    location 2 1       0           1     0                                        location 3 1       0           1     0                                        location 4 1       0           1     0                                        Average    1.0     0.0         1.0   0.0                                      ______________________________________                                    

EXAMPLE 7 Amylase Assay

The implants according to the invention are manufactured of starch whichis broken down quickly in the body. This example shows that a rapidinduction of the starch-degrading enzyme amylase takes place. Theamylase activity was determined in pig tissue after implantation of theimplants according to the invention and in untreated tissue as control.Tissue extracts of tissue homogenates were prepared in salt solution andquantified for amylase activity.

Tissue extracts were prepared at a temperature between 0° and 10° C.From three tissue samples and three controls a gram was cut into slicesand stored on ice. The tissue slices and 5 ml 0.9% sodium chloridesolution were mixed in a 10 ml tube of a Waring blender for 3 minutes at20,000 RPM. During this procedure the tube was cooled with an ice watermixture. The resulting suspension was collected in a clean tube. Theblender tube and the knife were washed with 4 ml salt solution which wasalso added to the suspension whereby the final volume became 10 ml.Finally, the suspension was centrifuged for 12 minutes at 4° C. and 1100RCF and the supernatant kept at -80° C. The assays of this tissueextract were performed on 1 to 10 diluted concentrations of tissueliquid components.

The amylase activity was determined according to Sigma procedure no.700. The procedure makes use of a colour reaction between starch andiodine which produces an intense blue colour, while the oligosaccharidesproduce a red colour as according to the diagram below. ##STR1## Thecolour change from blue to red-brown was sufficiently pronounced to makea visual detection of the end point possible without the use of aspectrophotometer. The end point was determined by removing parts of theserum starch reaction mixture at predetermined intervals and addingthereof to an iodine solution. As long as starch is present a purplishcolour develops. During the course of the incubation the colour changesfrom blue to blue-purple to red-purple and eventually to red-brown. Thisis the end point. The result is evaluated as follows:

0=no colour change; purplish

1=colour change; red-purple

2=end point; red-brown

The amylase activity in the sample is calculated by means of theformula: ##EQU1##

The results are shown in table 13.

                                      TABLE 13                                    __________________________________________________________________________    Incubation                                                                         Sample                   Sample                                          time                A    A                   B    B                           (min)                                                                              A    A    A    1:5  1:10 B    B    B    1:5  1:10                        __________________________________________________________________________     2   0    0    0    0    0    0    0    0    0    0                            4   0    0    1    0    0    0    0    0    0    0                            6   1    1    2    0    0    0    0    0    0    0                            8   2    2    2    0    0    0    0    0    0    0                           10   2    2    2    0    0    0    0    1    0    0                           12   2    2         0    0    1    1    2    0    0                           15                  0    0    2    2    2    0    0                           18                  0    0    2    2    2    0    0                           23                  1    0    2    2         0    0                           28                  2    0                   0    0                           32                  2    0                   0    0                           36                  2    0                   0    0                           44                       0                   0    0                           50                       0                   0    0                           56                       0                   1    0                           62                       1                   1    0                           70                       1                   2    0                           80                       2                   2    0                           90                       2                   2    0                           100                      2                        0                           110                                               1                           120                                               2                           Amylase/                                                                           2250 2250 3000 3200 2600 1200 1200 1500 1500 1500                        sample                                                                        (U/dl)                                                                        Average/                                                                           2660 U/dl                1380 U/dl                                       sample                                                                        __________________________________________________________________________

Normal serum levels of amylase activity in humans vary between 50 and200 U/dl. The recorded results demonstrate that the test is sensitive topig amylase and that normal tissue values (controls) are at least 10times as high as normal human serum values. The data likewise indicatesthat the amylase activity in pig tissue treated with starch is at leasttimes as high as in normal human serum. The result provides noinformation about the localization of the amylase, or whether it isintra- or extracellular and whether this is a local or systemicreaction. The conclusion is that implantation of starch implants in pigtissue causes a reaction characterized by an increased amylase activityin the tissue.

EXAMPLE 8 Tissue Reactions to Implantation of the Implant According tothe Invention in the Pig

In this experiment a pig receives implants at different times which areintroduced using the instrument described in the Netherlands patentapplication 92.00844.

A pig of about 65 kg received a first series of implants in the leftside of its body on day 1. Just before introduction the implants weresterilized in 70% alcohol for several seconds and dried in the air. Forinjection the tip of the barrel of the aforementioned instrument washeld about 1 cm from the skin, wherein the longitudinal axis waspositioned as perpendicularly of the skin as possible. The injections 1and 2 were given successively in the neck, injections 3, 4 and 5 in themusculus longissimus dorsi and the injections 6, 7 and 8 one belowanother in the buttock. The injection locations were disinfected usinggentian violet spray with chlortetracycline therein. After 18 days thewhole procedure was repeated on the right side of the pig. Three dayslater, on day 21, the pig was sacrificed by means of an injection of 50cc euthesate in the vena cava cranialis. The injection locations wereshaven and evaluated.

Very small scars were observed in the skin on the left side (after 21days). On the right side no swellings were visible but there were veryslight, small wounds with a small blood scab. Mild redness was onlyobserved at the eighth injection.

During dissection it was found that the injections 1 and 2 on the leftside left no trace whatever, subcutaneous or intramuscular. Injections 3and 4 are also no longer discernible subcutaneously, but intramuscularlyan elongate connective tissue strand of several centimeters length andless than one millimeter thickness is visible. Of the injections 5 to 8on the left side no trace could be found.

The injections on the right side (3 days) displayed at a depth of 2 cm aslight reaction with a cross-section of 1 cm wherein no swelling wasobserved but only a small dark discolouring. Injection 3 displayedsubcutaneously very light bleeding and mild oedema. At injection 4nothing was discernable subcutaneously while at 3 cm depthintramuscularly small bleeding with calcification granules was observed.The results of injection 5 corresponded with those of injection 3, whilefor the injections 6, 7 and 8 small bleedings were observedsubcutaneously and intramuscularly along the bullet passage to a depthof 5-6 cm.

On the microscopic coupes can be seen that three days after implantationa degeneration of still vital muscle tissue occurs, in addition to smalltemporary calcifications, very small bleeding caused by capillaryvessels which are torn by the projectile as it shoots past, necroticmaterial which is not vital and is probably crushed by the projectile asit passes, and cellular clearing reactions indicated by the presence oflymphocytes, polymononuclear cells and-macrophages. Because the tissuedestruction is very minimal there need be no fear of puss and/or fistulaformation and after a few weeks the clearing reactions will only leave aslight scar formation.

EXAMPLE 9 In Vivo Injection Test

In this experiment is determined whether the animals display abnormalactivity or appear to have a pain sensation. It is also determined whatthe effect of the injection is on the skin.

Seven male pigs with an average weight of 53 kg. were injected on day 1,20, 38, 62 (2×) and 67 with an implant according to the invention usingthe instrument as described in NL 92.00844.

For 7 days, following every injection and particularly after the firsthour and after 6 to 7 hours, a record was kept of all pigs as to whetherthe animals displayed abnormal activities or gave the impression ofbeing in pain. The injection locations were checked for bleeding,swelling, painfulness, redness or exudate after 1 hour, 6 to 7 hours, 1,2, 3, 4, 5, 6 and 7 days. On day 81 the animals were sacrificed and itwas visually determined whether the injections had left any trace.

From this experiment it was found that the administering of the implantshad no noticeable effect on the animals. Some animals displayed a mildswelling of the skin at the position of the injection but after 3 to 4days these phenomena had completely disappeared. After 81 days no tracewhatever of the total of 42 administered implants was found, nor wereany scars observed which could have been caused by the implants. It canbe concluded therefore that administering of the implants has no adverseeffect whatever on the animals.

EXAMPLE 10 The Relation Between Cytotoxicity and Processing

It was noted that, during injection moulding of the starch, productswhich stayed longer in the injection moulding machine had a brownercolour than other products. The brown colouring could be an indicationof cytotoxicity. In this example quantitative data are collectedrelating to the cytotoxic effects of the test material on a fibroblastmonolayer.

Four groups of samples were used. Their processing time in the machinewas linked to their brown colouring. The samples A, B and C representthree groups with an increase in brown coloration clearly discerniblewith the naked eye. A lubricant was necessary to enable removal of theseproducts from the mould. Sample D showed the least brown coloration, wasproduced in a sequential series and required no lubricant.

The classification of the samples A, B, C and D was further based onoptical density (OD), which was determined using a Kinetic Readerspectrophotometer (Biotek, model EL 312E). The optical density wasmeasured at 380 nm and 405 nm. The samples were cut into pieces ofseveral millimeters in length in order to obtain cylinders which werelaid on the bottom of a 96-well microtitre plate. The plate is placed inthe spectrophotometer. Table 16 shows the results.

The cytotoxicity was tested on the basis of a procedure according to theISO 10993-5 and USP XXII standards.

The samples were sterilized with ethanol and deaerated for more than 48hours prior to the test. All procedures were performed under sterileconditions.

Extracts were prepared by extracting quantities of test material andcontrol material with an outer surface area of 60 cm² for 24 hours at37° C. in 20 ml minimal essential medium (MEM, tissue culture mediumsupplemented with 10% foetal calf serum). USP neg. UHMW (Ultra HighMolecular Weight) polyethylene was used as negative control. Thepositive control was an RIVM-pos. Latex.

In summary, the procedure was as follows: a monolayer of human skinfibroblasts (PK*$) was cultured to 80-100% confluence and brought intocontact with an extract of the test material (n=3), the negative controlmaterial (n=3) or the positive control (n=1). After 24 hours of exposureto the extract at 37° C. the cell were examined and the cytotoxiceffects were determined microscopically by evaluation of:

a=interruption of the monolayer

b=degree of cellular lysis

c=change in cell morphology

The scores for a, b and c were corrected for the negative control, whichresults in the microscopic average. By treating the wells which wereused for a, b and c, with trypsin the cells were suspended, whereafterthey were counted microscopically with a Burker chamber. In this mannerthe inhibition of the cell proliferation (d) after 24 hours wasdetermined. A correction was made for the negative control.

The different observations were classified as shown in the followingtable 14.

                                      TABLE 14                                    __________________________________________________________________________    disruption of the                                                                         degree of cellular                                                                    changes in cell mor-                                                                     inhibition cell                                monolayer ML                                                                              lysis CL                                                                              phology CM growth CG                                      __________________________________________________________________________    class 0                                                                           none observed                                                                         none observed                                                                         no changes,                                                                               0-10%                                                             normal cells                                              class 1                                                                           slight  0-5%    slight changes, some                                                                     10-30%                                                             cells affected                                            class 2                                                                           mild     5-10%  mild changes, some                                                                       30-50%                                                             cells round and/or coil                                                       shaped                                                    class 3                                                                           moderate                                                                              10-20%  moderate changes                                                                         50-70%                                                             many cells round and/                                                         or coil shaped                                            class 4                                                                           serious >20%    serious changes, rough-                                                                   70-100%                                                           ly all cells show mor-                                                        phological changes                                        __________________________________________________________________________

Acceptance criteria are shown in table 15.

                  TABLE 15                                                        ______________________________________                                        cytotoxic response                                                                         cytotoxic effect                                                                          sufficient/insufficient                              ______________________________________                                        0-1          not toxic   sufficient                                           1-3          slightly  toxic                                                                           sufficient                                           3-5          mildly  toxic                                                                             test again                                           5-7          moderately toxic                                                                          insufficient                                         7-8          very  toxic insufficient                                         ______________________________________                                    

Table 16 shows the compilation of the test results.

                  TABLE 16                                                        ______________________________________                                                                        total cytotoxic                               Sample      OD.sub.380                                                                            OD.sub.405  response                                      ______________________________________                                        A           0.529   0.373       2.4 sufficient                                B           0.823   0.783       5.2 insufficient                              C           1.189   0.973       7.0 insufficient                              D           0.610   0.640       2.6 sufficient                                background  0.060   0.042                                                     ______________________________________                                    

The test shows a clear positive linear correlation between the browncolouring and the cytotoxicity of the material. The use of lubricant hasno effect on the total cytotoxicity response.

EXAMPLE 11 The Administering of an Implant by Means of Pushing

An implant according to the invention manufactured from starch which isdestructurized according to example 1 with a length of 17.5 mm, adiameter of 3.00 mm, a radius of the tip of 0.25 mm and an angle betweenthe tangent lines of the side walls of the tip of 60° was administeredsubcutaneously to a piglet and a calf. The implant was pushed into theauricle. The four signs of inflammation, redness, swelling, warmth andpain were not observed. Nor was any bleeding seen.

When an empty implant was introduced the cylindrical part haddisappeared within two hours, while the solid tip could no longer beseen after 5 hours. The sharp tip was no longer sharp after a fewseconds, whereby there was no danger of the skin being pierced frominside.

EXAMPLE 12 Adjuvant Action of the Implants

In order to test whether the implants according to the invention haveany effect in the administering of vaccinating substances, the modelinoculant bovine serum albumin (BSA) was administered in three differentways.

The first series of bullets contained a water-in-fat emulsion (withTween™ and Spans™) as adjuvant. The watery phase contained 500 μg BSA asantigen. The second series of bullets each contained a dry pellet with adiameter of 2 mm, consisting of a carrier substance (sugar) and 500 μgBSA. The third series of bullets was prepared according to the abovedescribed freezing method. Placed into each of these bullets was awatery solution with 500 μg BA which remained behind as powder in thebullets after freeze-drying. The bullets were sealed with a droplet offat.

The bullets with the known adjuvant were not found to give astatistically significantly higher immunological response than thebullets to which no adjuvant was added. It follows herefrom that thebullets themselves also have an adjuvant action.

I claim:
 1. A transdermal implant comprising substantially fullydestructurized starch wherein the implant is a bullet.
 2. Thetransdermal implant as claimed in claim 1 wherein the is hollow.
 3. Thetransdermal implant as claimed in claim 2 wherein the bullet furthercomprises a filling comprising at least one therapeutic, diagnostic orprophylactic compound and optionally a suitable diluent.
 4. Thetransdermal implant as claimed in claim 1 wherein the bullet is solid.5. The transdermal implant as claimed in claim 4 wherein the bulletcomprises a mixture of substantially destructurized starch and at leastone therapeutic, diagnostic or prophylactic compound.
 6. The transdermalimplant as claimed in claim 1 wherein the implant comprises a tip suchthat manual pushing of the implant through skin is possible.
 7. Thetransdermal implant as claimed in claim 6 wherein the radius r of thetip of the implant is smaller than 0.5 mm, and the angle which thetangent lines of the side surfaces of the tip form with each other isnot smaller than 30° and up to a maximum of 120° in order to enablepushing of the implant through skin of 0.1 to 1.5 mm thickness.
 8. Thetransdermal implant as claimed in claim 7 wherein the radius r of thetip of the implant is smaller than 0.25 mm and the angle which thetangent lines of the side surfaces of the tip form with each other isbetween 45° and 60°.
 9. The transdermal implant as claimed in claim 8,wherein the radius r of the tip of the implant is between 0.03 and 0.1mm.
 10. The transdermal implant as claimed in claim 8 wherein thefilling comprises an inoculant.
 11. The transdermal implant as claimedin claim 2 wherein the bullet contains an identification chip.
 12. Thetransdermal implant as claimed in claim 3 wherein the filling comprisesa therapeutic or prophylactic composition.
 13. A method of administeringan implant transdermally comprising introducing the transdermal implantof claim 1 under the skin.
 14. The method of claim 13 wherein thetransdermal implant comprises substantially fully destructurized starchand at least one therapeutic, diagnostic or prophylactic compound,optionally in combination with a suitable diluent.
 15. A method forfilling a hollow bullet transdermal implant with a therapeutic,diagnostic or prophylactic compound comprising freezing the implant,cooling a watery solution containing the therapeutic, diagnostic orprophylactic compound, pouring the watery solution into the implant,freezing the filled implant, and freeze drying the filled and frozenimplant.